PEG is a water-soluble, amphiphilic compound characterized by low immunogenicity, low antigenicity and low toxicity (J. M. Harris et al, In Poly(ethylene glycol) chemistry and biological applications, 1st edition, San Francisco, ACS, 1997; J. S. Kang et al, Expert Opin Emerg Drugs, 2009, 14, 363-380). PEGylation, the covalent attachment of PEG chains to proteins, has been used to advantage to improve the stability and pharmacokinetics of numerous biological therapeutics (Y. J. Wang, et al, J. Controlled Release, 2010, 145, 306-313; D. E. Borchmann, et al, Rapid Commun., 2014, 35, 27-43). In general, PEGylated molecules exhibit enhanced solubility and stability in vitro and in vivo and improved safety profiles relative to their unPEGylated counterparts. Further to the above, PEGylation has been shown to impart numerous clinical benefits to PEGylated molecules, including increased efficacy, decreased side effects, and lower frequency of dosing (J. Kling BioProcess International 2013, 11, 35-43). Since the U.S. Food and Drug Administration's (FDA's) first approval of a PEGylated product (ADAGEN®; pegademase bovine) in 1990, a dozen PEGylated products have been approved and many more are in development. The worldwide market for PEGylated proteins was estimated to be about $7 billion in 2012 and sales of PEGylated protein therapeutics are projected to outpace the biopharmaceutical market in years to come (J. Kling BioProcess International 2013, 11, 35-43).
Despite the clear advantages of PEGylated proteins, the PEGylation process is complicated and often results in low yields. The process also suffers from difficulties associated with the variable nature of PEG conjugations, both with regard to location and number on a protein. Such variability can in turn lead to deleterious alterations at binding sites or active sites on a PEGylated protein that can reduce biological activity thereof. Reaction mixtures also typically comprise various undesirable species, including PEGylated isoforms (positional isoforms), excessively PEGylated proteins, native (unPEGylated) proteins, and unreacted PEGs. Purification of the desired species of PEGylated proteins is thus complicated by variabilities in the PEGylation process. In light of the above, the PEGylation reaction has to be approached in a very protein/product specific fashion, which impairs development of generalized processes for both the reaction and purification of PEGylated proteins (Yoshimoto et al, Biotechnol. J., 2012, 7, 592-593; Fee et al, In Janson (Ed.), Protein Purification (3rd edition), John Wiley & Sons, Inc., Hoboken, 2011, 339-362).
In an effort to address some of the aforementioned challenges, methods are being developed to explore extrinsic and intrinsic chemical reactivity for site-selective bioconjugation (A. Dumas, et al, Angew. Chem. Int. Ed., 2013, 52, 3916-3921; N. Li, et al, J. Am. Chem. Soc., 2011, 133, 15316-15319; Z. Zhou, et al, Bioconj. Chem., 2014, 25, 138-146; M. Wendeler, et al, Bioconj. Chem., 2014, 25, 93-101; A. C. Obermeyer, et al, Angew. Chem. Int. Ed., 2014, 53, 1057-1061; Y.-M. Li, et al, Angew. Chem. Int. Ed., 2014, 126, 2230-2234; N. Toda, et al, Angew. Chem. Int. Ed., 2013, 52, 12592-12596; M. Marsac, et al, Bioconj. Chem., 2006, 17, 1492-1498). Particulars pertaining to extrinsic bioconjugation and intrinsic bioconjugation, which are one-step and two-step processes, respectively, are discussed in detail herein below. In short, although advances have been made using these approaches, neither approach appears well suited to the development of generalized processes for the efficient PEGylation of proteins.
Thus, there remains a need to develop new protocols that enable site-specific PEGylation of peptides and proteins.